Conjugates for delivery of biologically active compounds

Abstract

A construct suitable for delivery of a biologically active compound into cells, comprising: (d) a positively charged peptide; (e) a targeting-delivery peptide; and (f) the biologically active compound; wherein the positively charged peptide is covalently attached to the targeting-delivery peptide and the biologically active compound is covalently or non-covalently attached to the resultant chimeric cell delivery peptide.

1 . A construct suitable for delivery of a biologically active compound into cells, comprising:
 (a) a positively charged peptide;   (b) a targeting-delivery peptide; and   (c) the biologically active compound;   wherein the positively charged peptide is covalently attached to the targeting-delivery peptide and the biologically active compound is covalently or non-covalently attached to the resultant chimeric cell delivery peptide.   
     
     
         2 . A construct according to  claim 1  wherein the positively charged peptide is covalently attached to the amino terminus of the targeting-delivery peptide. 
     
     
         3 . A construct according to  claim 1  wherein the positively charged peptide is arginine rich. 
     
     
         4 . A construct according to  claim 1  wherein at least 20% of the amino acids in the positively charged peptide are arginine (R). 
     
     
         5 . A construct according to  claim 1  wherein the positively charged peptide comprises a sequence of the formula (RZR(Z) l (ILFQY) m ) n  or a functional derivative thereof, wherein Z is an aminoalkyl spacer, l is 0 or 1, m is 0 or 1 and n is from 2 to 6. 
     
     
         6 . A construct according to  claim 5  wherein Z is 6-aminohexanoyl (X) or betaalanyl (B). 
     
     
         7 . A construct according to  claim 5  wherein the positively charged peptide comprises any of the sequences selected from the group consisting of: SEQ ID NOS:1-44, RXRZRXR, RBRZRBR, RXRZRBR, RBRZRXR, RXRZRXRRXR(RXRZ-SEQ ID NO: 1), RXRRXRZRXR (SEQ ID NO: 1-ZRXR), RXRZRXRZRXR, RXRILFQYRXRZRXR (SEQ ID NO: 5-ZRXR), RXRZRXRILFQYRXR(RXRZ-SEQ ID NO: 5), RBRZRBRRBR(RBRZ-SEQ ID NO: 2), RBRRBRZRBR (SEQ ID NO: 2-ZRBR), RBRYRBRZRBR (SEQ ID NO: 22-ZRBR), RBRZRBRILFQYRBR(RBRZ-SEQ ID NO: 6), RBRILFQYRBRZRBR (SEQ ID NO: 6-ZRBR), RXRZRBRRXR(RXRZ-SEQ ID NO: 4), RXRRBRZRXR (SEQ ID NO: 4-ZRXR), RXRZRBRZRXR, RXRZRBRILFQYRXR(RXRZ-SEQ ID NO: 8), RXRILFQYRBRZRXR (SEQ ID NO: 7-ZRXR), RXRZRBRRBR(RXRZ-SEQ ID NO: 2), RXRRBRZRBR (SEQ ID NO: 3-ZRBR), RXRZRBRZRBR, RXRZRBRILFQYRBR(RXRZ-SEQ ID NO: 6), RXRILFQYRBRZRBR (SEQ ID NO: 7-ZRBR), RXRZRXRRBR(RXRZ-SEQ ID NO: 3), RXRRXRZRBR (SEQ ID NO: 1-ZRBR), RXRZRXRZRBR, RXRZRXRILFQYRBR(RXRZ-SEQ ID NO: 7), RXRILFQYRXRZRBR (SEQ ID NO: 5-ZRBR), RBRZRXRRBR(RBRZ-SEQ ID NO: 3), RBRRXRZRBR (SEQ ID NO: 4-ZRBR), RBRZRXRZRBR, RBRZRXRILFQYRBR (RBRZ-SEQ ID NO: 7), RBRILFQYRXRZRBR (SEQ ID NO: 8-ZRBR), RBRZRXRRXR (RBRZ-SEQ ID NO: 1), RBRRXRZRXR (SEQ ID NO: 4-ZRXR), RBRZRXRZRXR, RBRZRXRILFQYRXR(RBRZ-SEQ ID NO: 5), RBRILFQYRXRZRXR (SEQ ID NO: 8-ZRXR), RBRZRBRRXR(RBRZ-SEQ ID NO: 4), RBRRBRZRXR (SEQ ID NO: 2-ZRXR), RBRZRBRZRXR, RBRZRBRILFQYRXR(RBRZ-SEQ ID NO: 8), and RBRILFQYRBRZRXR (SEQ ID NO: 6-ZRXR), or a functional derivative thereof. 
     
     
         8 . A construct according to  claim 1  wherein the targeting-delivery peptide is selected from MSP, HSP, AAV6, AAV8 and TAT or a functional derivative thereof. 
     
     
         9 . A construct according to  claim 8  wherein the MSP peptide is ASSLNIA (SEQ ID NO: 45) or a functional derivative thereof, the HSP peptide is SKTFNTHPQSTP (SEQ ID NO: 46) or a functional derivative thereof, the AAV6 peptide is TVAVNLQSSSTDPATGDVHVM (SEQ ID NO: 47) or a functional derivative thereof, the AAV8 peptide is IVADNLQQQNTAPQIGTVNSQ (SEQ ID NO: 48) or a functional derivative thereof or the TAT peptide is YGRKKRRQRRRP (SEQ ID NO: 49) or a functional derivative thereof. 
     
     
         10 . A construct according to  claim 5  wherein the functional derivative is a polypeptide with a sequence which has homology to any of the specific sequences mentioned in  claims 5  to  9  and which is able to improve delivery of the compound into cells. 
     
     
         11 . A construct according to  claim 1  wherein the construct comprises the sequences shown in any of SEQ ID NOs: 50 to 54. 
     
     
         12 . A construct according to  claim 1  wherein the cells are cardiac muscle, skeletal muscle, smooth muscle or contractile cells. 
     
     
         13 . A construct according to  claim 1  wherein the biologically active compound comprises a nucleic acid, a DNA molecule, a peptide, a protein, a DNAzyme, a Ribozyme, a chromophore, a fluorophore, and/or a pharmaceutical. 
     
     
         14 . A construct according to  claim 13  wherein the nucleic acid comprises nucleic acid with phosphodiester, 2′O-methyl, 2′ methoxy-ethyl, phosphoramidate, methylphosphonate, and/or phosphorothioate backbone chemistry, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA), plasmid DNA or small interfering RNA (siRNA). 
     
     
         15 . A construct according to  claim 13  wherein the nucleic acid comprises a sequence capable of targeting a sequence responsible for exon skipping in a mutated pre-mRNA at an exon to be skipped or included, wherein inducing exon skipping or inclusion corrects the expression of said mutated pre-mRNA and wherein without correction the mutated pre-mRNA fails to express functional protein. 
     
     
         16 . A composition comprising the construct of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         17 - 21 . (canceled) 
     
     
         22 . A method of delivering a biologically active compound into a cell comprising contacting said cell with a construct according to  claim 1  comprising the biologically active compound. 
     
     
         23 . (canceled) 
     
     
         24 . A method according to  claim 22 , wherein the method is for treating or diagnosing a cardiac or skeletal muscle disease in a subject. 
     
     
         25 . A method according to  claim 24  wherein the skeletal muscle disease is a muscular dystrophy phenotype, optionally Duchenne muscular dystrophy (DMD). 
     
     
         26 . A method according to  claim 22  wherein the construct is administered by injection, optionally by intramuscular or intravenous injection.